Synergistic effects of mesenchymal stem cell-derived extracellular vesicles and dexamethasone on macrophage polarization under inflammatory conditions.

The undesirable inflammation and the excessive M1 macrophage activity may lead to inflammatory diseases. Corticosteroids and stem cell therapy are used in clinical practice to promote anti-inflammatory responses. However, this protocol has limitations and is associated with numerous side effects. In this study, the synergistic anti-inflammatory effects of dexamethasone (Dex) and mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) were evaluated to enhance the polarization of M1 inflammatory macrophages into the anti-inflammatory (M2) phenotype. Hence, we designed different combinations of Dex and EVs using three methods, including EVs isolated from Dex-preconditioned MSCs (Pre-Dex-EVs), EVs loaded with Dex (L-Dex-EVs), and EVs and Dex co-administration (Dex + EVs). All designed EVs had a significant effect on reducing the expression of M1-related genes (iNOS, Stat1, and IRF5), cytokines (IL6 and TNF-a), and CD markers (CD86) in lipopolysaccharide-stimulated macrophages. On the other hand, these combinations promoted the expression of alternative-activated M2-related genes (Arg-1, Stat6, and IRF4), cytokine (IL10), and CD markers (CD206).The combination of Dex and MSC-EVs enhances the effectiveness of both and synergistically promotes the conversion of inflammatory macrophages into an anti-inflammatory state.

Avastin-Loaded 3D-Printed Alginate Scaffold as an Effective Antiadhesive Barrier to Prevent Postsurgical Adhesion Bands Formation.

Postoperative adhesion can cause complications, such as pain and organ blockage, in the abdominal regions. To address this issue, surgical techniques and antiadhesive treatments are applied. Given the significant role of vascularization in adhesion band formation, Avastin (Ava) that targets vascular endothelial growth factor (VEGF) can be applied to prevent peritoneal adhesion bands. Moreover, Alginate (Alg), a natural polysaccharide, is a promising physical barrier to prevent adhesion bands. Incorporating Ava into Alg hydrogel in a form of 3D-printed scaffold (Alg/Ava) has potential to suppress inflammation and angiogenesis, leading to reduce peritoneal adhesion bands. Following physical, morphological, and biocompatibility evaluations, the efficacy of Alg and Ava alone and their combination in Alg/Ava on the formation of postsurgical adhesions is evaluated. Upon confirming physical stability and sustained release of Ava, the Alg/Ava scaffold effectively diminishes both the extent and strength of adhesion bands. Histopathological examination shows that the reduction in fibrosis and inflammation is responsible for preventing adhesion bands by the Alg/Ava scaffold. Additionally, the cytokine assessment reveals that this is due to the inhibition in the secretion of VEGF and Interleukin 6 suppressing vascularization and inflammatory pathways. This study suggests that a 3D-printed Alg/Ava scaffold has great potential to prevent the postsurgical adhesion bands.

Lanthanide complexes facilitate wound healing by promoting fibroblast viability, migration and M2 macrophage polarization.

A tridentate ligand LH3 ((2-hydroxy-3-methoxybenzylidene)-2-(hydroxyimino)propanehydrazide) comprising o-vanillin, hydrazone and oxime donor groups has been employed to prepare a series of tetranuclear Ln(III) complexes. The reaction of ligand LH3 with Ln(NO3)3 [Ln = Sm, Eu, Gd, Tb, Dy, Ho, Er] in MeOH yielded Ln4(LH)6(MeOH)2 (Ln = Sm(1), Eu(2), Gd(3), Tb(4), Ho (6) and Er (7))] whereas the corresponding reaction with Dy(NO3)3 afforded Dy4(LH)4(LH2)2(OH)2 (5). All complexes were characterized by various analytical techniques including single crystal X-ray diffraction, IR spectroscopy, UV-Vis spectroscopy, and elemental analysis. To investigate the potential of these lanthanide complexes for wound healing applications, their effects on fibroblast viability, migration, and M2 macrophage polarization were evaluated. The cytotoxicity assessment revealed that complexes 2(Eu), 4(Tb), 5(Dy), and 7(Er) significantly enhanced fibroblast viability compared to the negative control (NC). In vitro wound healing assay demonstrated that complexes 2(Eu) and 7(Eu) substantially promoted fibroblast migration compared to the NC. Moreover, complex 2(Eu) exhibited significant anti-inflammatory effects by reducing the phagocytic ability of lipopolysaccharide (LPS)-stimulated macrophage cells and attenuating nitric oxide (NO) production. In conclusion, among the series of complexes tested, complex 2(Eu) displayed the most potent anti-inflammatory effect on macrophage cells, while simultaneously promoting fibroblast viability and migration. This unique combination of properties renders complex 2 (Eu) highly promising for wound healing applications.

BACTERIOPHAGE M13 MODULATES THE SEPSIS-RELATED INFLAMMATORY RESPONSES AND ORGAN DAMAGE IN A CLP MODEL.

ABSTRACT: Background: Sepsis is a life-threatening disorder that leads to the induction of inflammatory responses and organ failure. Phage therapy is a new approach to controlling infections resistant to common treatments, including sepsis. Several studies have shown the effect of lytic bacteriophages on infection control by reducing the bacterial load. The present study deals with lysogenic bacteriophage M13 on the inflammatory responses caused by cecal ligation and puncture (CLP)-induced sepsis in a mouse model. Methods Bacteriophage M13 harvested from ER2738, titrated, and confirmed by transmission electron microscopy analysis. In vitro toxicity and immunomodulatory effect of bacteriophage M13 were assessed on splenocytes by measurement of cell viability and the production level of cytokines, nitric oxide, and reactive oxygen species. For in vivo experiments, 8-weeks-old male C57BL/6 mice were randomly divided into the following three groups: CLP + NS (treated with normal saline), CLP + M13 (treated with an intraperitoneal injection of 10 9 PFU/mL of bacteriophage M13), and sham + NS (induced surgery but without ligation and puncture, treated with NS). The mice were killed at different time points after surgery (6, 24, 48, and 72, n = 10 for each time point of each group). The kidney, liver, and lungs were harvested for histopathological analysis, and blood was obtained for cytokine and liver enzyme assay. The spleen was used to assess the bacterial load using colony-forming unit assay. The rectal temperature and survival were evaluated during the study. Results According to the in vitro results, 10 9 PFU/mL of bacteriophage M13 was not toxic and did not affect the level of cytokine, nitric oxide, and reactive oxygen species production by splenocytes, but it reduced the inflammatory response of splenocytes in responses to LPS. In vivo studies indicated that the amount of proinflammatory cytokines, liver enzymes, bacterial load, and organ failure were decreased in the CLP + M13 group compared with CLP + NS, whereas the survival rate was increased. Conclusions These experiments demonstrated that bacteriophage M13 could lessen the consequences related to sepsis in CLP mice and can be considered a therapeutic approach in sepsis.

Selective anti-cancer activity against melanoma cells using 3-O-acetyl-ß-boswellic acid-loaded 3D-Printed scaffold.

This study aimed to develop a local 3 D-printed bioactive graft using poly-caprolacton (PCL) as a drug carrier and 3-O-acetyl-ß-boswellic acid (ß-ABA) as an anticancer compound. ß-ABA-loaded 3 D-printed scaffold was fabricated and physically characterized. The results indicated more desirable mechanical and physical properties of the ß-ABA-loaded PCL mat in comparison with the PCL scaffold. Following sustained release of ß-ABA, the ß-ABA-loaded PCL scaffold revealed selective cytotoxic activity against melanoma cells, while the PCL + ABA with the bolus delivery of ß-ABA was toxic against fibroblast cells. Followed by the induction of apoptosis in melanoma cells at the gene level, the result of the western blot showed that the ß-ABA-loaded scaffold significantly up-regulated P53 and down-regulated BCL2, with an increment in the ratio of Bax/BCL2. The selective anti-cancer properties of ß-ABA-loaded 3 D printed scaffold against melanoma cells indicated that this scaffold could be potentially used as a bioactive graft to improve the melanoma treatment.

Comparative study of the cytotoxicity, apoptotic, and epigenetic effects of Boswellic acid derivatives on breast cancer.

This study aimed to compare the effect of Boswellic acid derivatives on the viability, apoptosis, and epigenomic profiling of breast cancer. According to the viability assays, 3-O-acetyl-11-keto-ß-Boswellic acid (AKBA) showed more toxicity against MDA-MB-231 cells when compared with the 3-O-acetyl-ß-Boswellic acid (ABA). In contrast, ABA revealed less toxicity against MCF-10A. Cell cycle and apoptosis assays determined the maximum apoptotic effect of AKBA on MCF-7, and MDA-MB-231 cells. Interestingly, ß-Boswellic acid (BA) and ABA did not promote the apoptosis in MCF-10A cells. Transwell migration assay indicated the greatest normalized inhibition (around 160%) in the migration of MDA-MB-231 cells induced by AKBA. The expression of P53, BAX, and BCL2 genes in cancerous cell lines has affirmed that both AKBA and ABA could induce the maximal apoptosis. Western-blot investigation demonstrated that the maximum over-expression of P53 protein (1.96 times) was caused by AKBA in MDA-MB-231 cells, followed by ABA in MCF-7 cells. The BCL2 protein expression was in agreement with the previously reported results. The global DNA methylation in both cancerous cells was reduced by ABA. These results suggest that ABA represented more epigenetic modulatory effect while AKBA shows more cytotoxic and apoptotic effect against breast cancer cell lines.

Coexistence of sickle cell disease and systemic lupus erythematosus is associated with quantitative and qualitative impairments in circulating regulatory B cells.

The incidence of connective tissue diseases such as systemic lupus erythematous (SLE), in adult patients with sickle cell disease (SCD), appears to be increasing. The exact causes underlying this increased risk are still unknown, but a link with B regulatory (Breg) cells is possible as these cells suppress inflammatory responses, and maintain tolerance. Quantitative and qualitative analyses of circulating Breg cells were performed in a cohort of SCD patients with SLE, and their levels were correlated with key soluble mediators promoting autoreactive B cells. We demonstrated that levels of Breg cells were significantly decreased in SCD patients with SLE compared to patients with SCD only or healthy controls. Functional analysis of Breg cells from SCD patients with SLE revealed impairments in IL-10 production that correlated with lower levels of STAT3 phosphorylation, without abnormal expression of IL-10 receptor on Breg cells. On the other hand, BAFF levels were substantially elevated in SCD patients with SLE, but not significantly associated with Breg cell levels. Collectively, these results indicated numerical and functional deficits of Breg cells in SCD patients with SLE and their capacity to maintain tolerance and control inflammation is imbalanced, which leads to the development of autoimmune responses.

Regulatory Non-Coding RNAs in Familial Hypercholesterolemia, Theranostic Applications.

Familial hypercholesterolemia (FH) is a common monogenic disease which is associated with high serum levels of low-density lipoprotein cholesterol (LDL-C) and leads to atherosclerosis and cardiovascular disease (CVD). Early diagnosis and effective treatment strategy can significantly improve prognosis. Recently, non-coding RNAs (ncRNAs) have emerged as novel biomarkers for the diagnosis and innovative targets for therapeutics. Non-coding RNAs have essential roles in the regulation of LDL-C homeostasis, suggesting that manipulation and regulating ncRNAs could be a promising theranostic approach to ameliorate clinical complications of FH, particularly cardiovascular disease. In this review, we briefly discussed the mechanisms and pathophysiology of FH and novel therapeutic strategies for the treatment of FH. Moreover, the theranostic effects of different non-coding RNAs for the treatment and diagnosis of FH were highlighted. Finally, the advantages and disadvantages of ncRNA-based therapies vs. conventional therapies were discussed.

Microscopic Evaluation of Ovarian Surface Epithelium Following Treatment with Conjugated Estrogens in a Mouse Model.

BACKGROUND: This study was designed to evaluate the effect of different concentrations of conjugated equine estrogens (CEE) on the ovarian epithelium of female CD1 mice. METHODS: Twenty-four female mice at 7 months with irregular estrus cycles were randomly divided into four groups of 6 mice each. Group one was considered as a control group and received a daily dose of 0.5ml of propylene glycol, for three weeks, while those in the treatment groups received a daily dose of 14µg/kg, 28µg/kg and 56µg/kg conjugated equine estrogens, respectively. RESULTS: The results from this study showed a strong correlation between elevated concentrations of CEE and histological changes in ovarian surface epithelium (OSE). They also showed that administration of high-dose estrogen created the conditions for excessive proliferation of OSE which may progress into the development of cysts in the ovaries. CONCLUSION: This study concluded that high concentrations of CEE may increase the chances of developing epithelial ovarian cancer.

Prevention of post-surgical adhesion bands by local administration of frankincense n-hexane extract.

Background: and purpose: The formation of postoperative intra-abdominal adhesion band formation may lead to severe complications. This study aimed to evaluate the preventive effect of local administration of frankincense n-hexane extract (FHE) on the formation of postsurgical adhesion bands. Materials and methods: FHE was extracted from the resin of a Boswellia sacra tree and its components were identified by gas chromatography-mass spectrometry (GC-MS). In an animal model, the expression levels of TNF-α and TGF-ß1 cytokines after application of FHE were assessed to check the inflammatory and fibrotic cues, respectively. Results: Following FHE compound analysis, in vivo experiments demonstrated that intraoperative local administration of FHE resulted in the prevention of adhesion band formation. The adhesion grades in the FHE-treated group were significantly lower than those in the negative control (NC) and the positive control (Interceed). The infiltration of inflammatory cells observed by histopathology revealed a significant anti-inflammatory potential of FHE. Furthermore, the gene expression results proved that significant suppression of TNF-α and TGF-ß1 was responsible for its antiadhesion properties. Conclusions: The study reported the potential of FHE as an ointment for the prevention of adhesion bands.

Evaluation of the solubility of 11-keto-ß-boswellic acid and its histological effect on the diabetic mice liver using a novel technique.

BACKGROUND AND AIM: The literature is scant on the effect of 11-keto-ß-boswellic acid (KBA) on the liver of diabetes-induced mice. This study was designed to develop a rapid, sensitive, accurate, and inexpensive detection technique for evaluating the solubility of KBA obtained from the gum resin of Omani frankincense (Boswellia sacra) in the liver of streptozotocin-induced diabetic mice using Fourier transform infrared (FTIR) reflectance spectroscopy coupled with principal components analysis (PCA). It also aimed to investigate the effect of KBA on histological changes in the hepatocytes of diabetic mice. MATERIALS AND METHODS: Eighteen mice were assigned to the healthy control group, the diabetic control group, or the KBA-treated diabetic group. Liver tissue samples from all groups were scanned using an FTIR reflectance spectrophotometer in reflection mode. FTIR reflectance spectra were collected in the wavenumber range of 400-4000 cm-1 using an attenuated total reflectance apparatus. RESULTS: FTIR reflectance spectra were analyzed using PCA. The PCA score plot, which is an exploratory multivariate data set, revealed complete segregation among the three groups' liver samples based on changes in the variation of wavenumber position in the FTIR reflectance spectra, which indicated a clear effect of KBA solubility on treatments. Histological analysis showed an improvement in the liver tissues, with normal structures of hepatocytes exhibiting mild vacuolation in their cytoplasm. CONCLUSION: KBA improved the morphology of liver tissues in the diabetic mice and led to complete recovery of the damage observed in the diabetic control group. FTIR reflectance spectroscopy coupled with PCA could be deployed as a rapid, low-cost, and non-destructive detection method for evaluating treatment effects in diseased liver tissue based on the solubility of KBA.

An engineered microfluidic blood-brain barrier model to evaluate the anti-metastatic activity of ß-boswellic acid.

BACKGROUND: The development of anti-cancer drugs with the ability to inhibit brain metastasis through the blood-brain barrier (BBB) is substantially limited due to the lack of reliable in vitro models. MAIN METHODS: In this study, the Geltrex-based Transwell and microfluidic BBB models were applied to screen the effect of ß-boswellic acid (ß-BA) on the metastasis of MDA-MB-231 cells through the BBB in static and dynamic conditions, respectively. MAJOR RESULTS: The toxicity assay revealed that ß-BA deteriorates MDA-MB-231 cells, while ß-BA had no detectable toxic effects on human umbilical vein endothelial cells (HUVECs) and astrocytes. Trans-endothelial electrical resistance evaluation showed sustainable barrier integrity upon treatment with ß-BA. Vimentin expression in HUVECs, evaluated using western blot, confirmed superior barrier integrity in the presence of ß-BA. The obtained results were confirmed using an invasion study with a cell tracker and a scanning electron microscope. ß-BA significantly inhibited metastasis by 85%, while cisplatin (Cis), a positive control, inhibited cancer cell migration by 12% under static conditions. Upon applying a dynamic BBB model, it was revealed that ß-BA-mediated metastasis inhibition was significantly higher than that mediated by Cis. CONCLUSIONS AND IMPLICATIONS:  In summary, the current study proved the anti-metastatic potential of ß-BA in both static and dynamic BBB models.

A competitive nature-derived multilayered scaffold based on chitosan and alginate, for full-thickness wound healing.

The aim of this study was to evaluate a bioactive multilayer wound dressing, based on chitosan and alginate. To enhance healing potential, Dracaena Cinnabari and Aloe Vera were loaded as separate layers into the scaffold. The bare and bioactive multilayered scaffolds were fabricated by an iterative layering freeze-drying technique. Following of topographical, chemical, and physical assessment, the performance of the scaffolds was evaluated in vitro and in vivo. The results revealed adequate attachment, and proliferation of human foreskin fibroblasts, indicating excellent biocompatibility of the bioactive scaffold. In vivo, the performance of the multi-layered scaffold loaded with the bioactive materials was comparable with Comfeel plus®. The wounds treated with the bioactive scaffold exhibited superior hypergranulation, fibroblast maturation, epithelization, and collagen deposition, with minimal inflammation, and crust formation. It is concluded that the synergism of extracellular matrix-mimicking multi-layered scaffolding with Aloe Vera and Dracaena Cinnabari could be considered as a supportive wound dressing.

Effects of prenatal tobacco smoke on cerebellum histomorphological changes at critical developmental stages in CD-1 mice / Efectos del humo de tabaco en los cambios histomorfológicos del cerebelo en las etapas críticas del desarrollo prenatal en ratones CD-1

SUMMARY: In this study the consequences of prenatal exposure to tobacco smokes on the histo-morphological changes of cerebellum was assessed by comparing the smoker mice to the nonsmoker mice. A total of 30 pregnant cd-1 mice were divided into three groups of 10 mice each and with two replicates per group (5 mice each). Following acclimation for five days, the mice were placed in a special modified smoking machine for 2 hours per day over a two- and three-week period for group two and group three, respectively. Group one was considered as a control group. Mice in the control group were exposed simultaneously to fresh air from the room, while those in the treatment groups were exposed to tobacco smoke from six commercial filter cigarettes, containing 0.8 mg of nicotine, 10 mg of tar, and 10 mg of carbon monoxide, for three 1-hour exposure periods every day for three weeks. The mice in the control group were exposed to room air for three 1-hour periods every day for the same period of three weeks. The results from this study showed a correlation between maternal smoking and histological changes in Neuron purkinjense (Purkinje cells) of the cerebellum. They also showed that prenatal smoking period may have caused more damage in the histology and structure of Neuron purkinjense in some juvenile mice. An increased incidence of morphology damage of the cerebellum's Neuron purkinjense' structures was also observed in fetuses with prolonged exposure to tobacco smoking. Exposure of in utero maternal smoking may interfere with brain biological development parameters, giving rise to structural abnormalities of the cerebellum. This study concluded that tobacco smoke exposure to pregnant mice may affect neurodevelopment which may induce behavioural changes as a result of reduced cerebellar size and function.

RESUMEN: Se evaluaron los efectos producidos por la exposición prenatal al humo de tabaco en ratones expuestos y no expuestos y los cambios histomorfológicos observados en el cerebelo en ambos grupos. Un total de 30 ratones cd-1 preñados se dividieron en tres grupos de 10 ratones cada uno y con dos réplicas por grupo (5 ratones cada uno). Después de la aclimatación durante cinco días, los ratones se colocaron en una máquina de fumar modificada, especial durante 2 horas al día, durante un período de dos y tres semanas para el grupo dos y el grupo tres, respectivamente. El grupo uno se consideró como grupo control. Los ratones del grupo de control fueron expuestos simultáneamente al aire limpio de la habitación, mientras que los grupos de tratamiento fueron expuestos al humo de tabaco de seis cigarrillos comerciales, que contenían 0,8 mg de nicotina, 10 mg de alquitrán y 10 mg de monóxido de carbono. durante tres períodos de 1 hora diariamente, durante tres semanas. Los ratones del grupo de control se expusieron al aire ambiente durante tres períodos de 1 hora todos los días durante el mismo período de tres semanas. Los resultados de este estudio mostraron una correlación entre el tabaquismo materno y los cambios histológicos en las neuronas purkinjenses (células de Purkinje). Se observó además que el período de tabaquismo prenatal puede haber causado mayor daño en la histología y estructura de las neuronas purkinjenses en algunos ratones jóvenes. También se observó una mayor incidencia de daño morfológico de las estructuras de las neuronas purkinjenses del cerebelo en fetos con exposición prolongada al tabaquismo. La exposición al tabaquismo materno en el útero puede interferir con los parámetros de desarrollo biológico del cerebro, dando lugar a anomalías estructurales del cerebelo. Este estudio concluyó que la exposición al humo del tabaco en ratones preñados puede afectar el desarrollo neurológico, lo que puede inducir cambios de comportamiento como resultado de la reducción del tamaño y la función del cerebelo.

New derivatives of 11-keto-ß-boswellic acid (KBA) induce apoptosis in breast and prostate cancers cells.

A series of new 11-keto-ß-boswellic acid were partially-synthesized by modifying the hydroxyl and carboxylic acid functional groups of ring A. The structures of the new analogs were confirmed by detailed spectral data analysis. Compounds 4, 5 and 9 exhibited potent anti-cancer results against two human tumor cancer cell lines having IC50 value of MCF-7 (breast) and LNCaP (prostate): 123.6, 9.6 and 88.94 µM and 9.6, 44.12 and 12.03 µM, respectively. Additionally, a maximum nuclear fragmentation was observed for 4 (78.44%) in AKBA treated cells after 24 hr followed by 5 and 9 with (74.25 and 66.9% respectively). This study suggests that the presence of hydrazone functionality (4 and 9) has effectively improved the potency of AKBA. Interestingly, compound 5 with a lost carboxylic acid group of ring A showed comparable potent activity. Highly selective AKBA requires further modification to improve its bioavailability and solubility inside the cancer cells.

Secondary metabolites from acridocarpus orientalis inhibits 4T1 cells and promotes mesenchymal stem cells (MSCs) proliferation.

Among medicinal plants, Acridocarpus orientalis (AO) possesses a remarkable anti-cancer potential, possibly because of its anti-oxidant property. In this study, the leaf and stem extracts from AO were assessed to find the bioactive compound with selective anti-cancer properties. The MTT viability and live and dead assays revealed that around 80% and 98% of 4T1 cells survival were declined after 48 h incubation with leaf and stem extracts, respectively. The leaf extract increased stem cell proliferation by 20% whereas the stem extract inhibited around 22% of stem cells proliferation after 48 h treatment. The live and dead assay of MSCs confirmed that 40% of the MSCs died when treated with AO stem extract. On the other hand, there were no dead cells after two days of treatment with the leaf extract. Followed by the induction of cell cycle arrest in G0/G1-phase, the real-time PCR demonstrated apoptosis properties in 4T1 cells through overexpression of Bax and down-regulation of BCL2 genes. Interestingly, within the pure compounds isolated from AO leaf extract, Morin was responsible for the inhibition of 4T1 cells proliferation as well as MSCs expansion, predicting to play an essential role in the treatment of cancer. The promising in vitro anti-cancer and stem cell-inductive properties of morin isolated from AO extract may provide a great potential to produce selective herbal derived drugs.

Accuracy of Platelet Counting by Optical and Impedance Methods in Patients with Thrombocytopaenia and Microcytosis.

OBJECTIVES: Obtaining accurate platelet counts in microcytic blood samples is challenging, even with the most reliable automated haematology analysers. The CELL-DYN(™) Sapphire (Abbott Laboratories, Chicago, Illinois, USA) analyser uses both optical density and electronic impedance methods for platelet counting. This study aimed to evaluate the accuracy of optical density and electrical impedance methods in determining true platelet counts in thrombocytopaenic samples with microcytosis as defined by low mean corpuscular volume (MCV) of red blood cells. Additionally, the impact of microcytosis on platelet count accuracy was evaluated. METHODS: This study was carried out between February and December 2014 at the Haematology Laboratory of the Sultan Qaboos University Hospital in Muscat, Oman. Blood samples were collected and analysed from 189 patients with thrombocytopaenia and MCV values of <76 femtolitres. Platelet counts were tested using both optical and impedance methods. Stained peripheral blood films for each sample were then reviewed as a reference method to confirm platelet counts. RESULTS: The platelet counts estimated by the impedance method were on average 30% higher than those estimated by the optical method (P <0.001). The estimated intraclass correlation coefficient was 0.52 (95% confidence interval: 0.41-0.62), indicating moderate reliability between the methods. The degree of agreement between methods ranged from -85.5 to 24.3 with an estimated bias of -30, suggesting that these methods generate different platelet results. CONCLUSION: The impedance method significantly overestimated platelet counts in microcytic and thrombocytopaenic blood samples. Further attention is therefore needed to improve the accuracy of platelet counts, particularly for patients with conditions associated with microcytosis.

The role of programmed cell death ligand-1 (PD-L1/CD274) in the development of graft versus host disease.

Programmed cell death ligand-1 (PD-L1/CD274) is an immunomodulatory molecule involved in cancer and complications of bone marrow transplantation, such as graft rejection and graft-versus-host disease. The present study was designed to assess the dynamic expression of this molecule after hematopoietic stem cell transplantation in relation to acute graft-versus-host disease. Female BALB/c mice were conditioned with busulfan and cyclophosphamide and transplanted with either syngeneic or allogeneic (male C57BL/6 mice) bone marrow and splenic cells. The expression of PD-L1 was evaluated at different time points employing qPCR, western blot and immunohistochemistry. Allogeneic- but not syngeneic-transplanted animals exhibited a marked up-regulation of PD-L1 expression in the muscle and kidney, but not the liver, at days 5 and 7 post transplantation. In mice transplanted with allogeneic bone marrow cells, the enhanced expression of PD-L1 was associated with high serum levels of IFNγ and TNFα at corresponding intervals. Our findings demonstrate that PD-L1 is differently induced and expressed after allogeneic transplantation than it is after syngeneic transplantation, and that it is in favor of target rather than non-target organs at the early stages of acute graft-versus-host disease. This is the first study to correlate the dynamics of PD-L1 at the gene-, protein- and activity levels with the early development of acute graft-versus-host disease. Our results suggest that the higher expression of PD-L1 in the muscle and kidney (non-target tissues) plays a protective role in skeletal muscle during acute graft-versus-host disease.

Busulphan-cyclophosphamide cause endothelial injury, remodeling of resistance arteries and enhanced expression of endothelial nitric oxide synthase.

Stem cell transplantation (SCT) is a curative treatment for malignant and non malignant diseases. However, transplantation-related complications including cardiovascular disease deteriorate the clinical outcome and quality of life. We have investigated the acute effects of conditioning regimen on the pharmacology, physiology and structure of large elastic arteries and small resistance-sized arteries in a SCT mouse model. Mesenteric resistance arteries and aorta were dissected from Balb/c mice conditioned with busulphan (Bu) and cyclophosphamide (Cy). In vitro isometric force development and pharmacology, in combination with RT-PCR, Western blotting and electron microscopy were used to study vascular properties. Compared with controls, mesenteric resistance arteries from the Bu-Cy group had larger internal circumference, showed enhanced endothelium mediated relaxation and increased expression of endothelial nitric oxide synthase (eNOS). Bu-Cy treated animals had lower mean blood pressure and signs of endothelial injury. Aortas of treated animals had a higher reactivity to noradrenaline. We conclude that short-term consequences of Bu-Cy treatment divergently affect large and small arteries of the cardiovascular system. The increased noradrenaline reactivity of large elastic arteries was not associated with increased blood pressure at rest. Instead, Bu-Cy treatment lowered blood pressure via augmented microvascular endothelial dependent relaxation, increased expression of vascular eNOS and remodeling toward a larger lumen. The changes in the properties of resistance arteries can be associated with direct effects of the compounds on vascular wall or possibly indirectly induced via altered translational activity associated with the reduced hematocrit and shear stress. This study contributes to understanding the mechanisms that underlie the early effects of conditioning regimen on resistance arteries and may help in designing further investigations to understand the late effects on vascular system.

Dynamics of early histopathological changes in GVHD after busulphan/cyclophosphamide conditioning regimen.

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for otherwise incurable diseases. Conditioning regimen is an important part of HSCT and consists of chemotherapy with or without irradiation. Conditioning exerts myelosuppressive, immunosuppressive and antitumor effects, but also contributes to HSCT-related complications including graft-versus-host disease (GVHD). Since almost 50% of the transplanted patients are conditioned with cytostatics without irradiation, we developed and characterized a GVHD mouse model following conditioning with busulphan and cyclophosphamide. Recipient Balb/c female mice were treated with busulphan (20 mg/kg/day for 4 days) and cyclophosphamide (100 mg/kg/day for two days). After one day of rest, recipient mice were transplanted with 2×10(7) bone marrow and 3×10(7) spleen cells from male C57BL/6 (allogeneic group) or female Balb/c (syngeneic/control group) mice. The allogeneic, but not syngeneic transplanted mice developed GVHD. Histopathology of the major internal organs (liver, pancreas, spleen, lungs, heart and kidney) was examined before conditioning start, after conditioning's end and 5, 7 and 21 days after transplantation using hematoxylin-eosin staining. Decreased spleen cellularity and diminished glycogen content in the liver were observed after conditioning regimen. Histopathological changes such as vasculitis, inflammation and apoptotic cell forms in liver, spleen, pancreas, lungs and heart were observed in allogeneic transplanted mice, however, only hypocellular spleen and extramedullar hematopoiesis were detected in syngeneic transplanted animals. No morphological changes were observed in kidney in either HSCT setting. This is the first study describing early histopathological changes after conditioning regimen with busulphan/cyclophosphamide and dynamics of GVHD development in several major internal organs.